Low-dose methotrexate in dermatology: the utility of serological monitoring in a real-world cohort.

When prescribing low-dose methotrexate, frequent serological testing is recommended in the dermatologic literature, although much of the supporting data is extrapolated from non-dermatologic conditions. We performed a retrospective cohort study to determine the cumulative incidence and timing of low-dose methotrexate-associated serological abnormalities over the first year of therapy, in a pragmatic cohort of patients with dermatologic compared to non-dermatologic diagnoses. Laboratory values recorded included white blood cell count, hemoglobin, platelet count, estimated glomerular filtration rate, alanine aminotransferase, and aspartate aminotransferase. Among 1376 patients, there were no cases of methotrexate-associated grade 4/very severe lab abnormality or fatality. Baseline risk factors associated with moderate-to-severe lab abnormalities included non-dermatologic diagnoses, low hemoglobin, low estimated glomerular filtration rate, and elevated transaminases. The incidence of moderate-to-severe lab abnormalities was 4.4% among all patients, 3.1% among patients with dermatologic diagnoses, and 2.3% among patients with normal baseline lab values. Lab abnormalities led to discontinuation of therapy in 0.8% of patients. Serious changes did not occur in the first two weeks of therapy. We conclude that the cumulative incidence of low-dose methotrexate-associated lab abnormality was lower in patients with dermatologic diagnoses or normal baseline testing and these factors may be used to adjust monitoring practices.

Protein structure prediction.

Protein structure prediction has matured over the past few years to the point that even fully automated methods can provide reasonably accurate three-dimensional models of protein structures. However, until now it has not been possible to develop programs able to perform as well as human experts, who are still capable of systematically producing better models than automated servers. Although the precise details of protein structure prediction procedures are different for virtually every protein, this chapter describes a generic procedure to obtain a three-dimensional protein model starting from the amino acid sequence. This procedure takes advantage both of programs and servers that have been shown to perform best in blind tests and of the current knowledge about evolutionary relationships between proteins, gained from detailed analyses of protein sequence, structure, and functional data.

Structural studies of the Nudix hydrolase DR1025 from Deinococcus radiodurans and its ligand complexes.

We have determined the crystal structure, at 1.4A, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked beta-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap(4)A (both at 1.6A resolution). In the Ap(4)A co-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.

Sequence conservation in families whose members have little or no sequence similarity: the four-helical cytokines and cytochromes.

Proteins for which there are good structural, functional and genetic similarities that imply a common evolutionary origin, can have sequences whose similarities are low or undetectable by conventional sequence comparison procedures. Do these proteins have sequence conservation beyond the simple conservation of hydrophobic and hydrophilic character at specific sites and if they do what is its nature? To answer these questions we have analysed the structures and sequences of two superfamilies: the four-helical cytokines and cytochromes c'-b(562). Members of these superfamilies have sequence similarities that are either very low or not detectable. The cytokine superfamily has within it a long chain family and a short chain family. The sequences of known representative structures of the two families were aligned using structural information. From these alignments we identified the regions that conserve the same main-chain conformation: the common core (CC). For members of the same family, the CC comprises some 50% of the individual structures; for the combination of both families it is 30%. We added homologous sequences to the structural alignment. Analysis of the residues occurring at sites within the CCs showed that 30% have little or no conservation, whereas about 40% conserve the polar/neutral or hydrophobic/neutral character of their residues. The remaining 30% conserve hydrophobic residues with strong or medium limitations on their volume variations. Almost all of these residues are found at sites that form the "buried spine" of each helix (at sites i, i+3, i+7, i+10, etc., or i, i+4, i+7, i+11, etc.) and they pack together at the centre of each structure to give a pattern of residue-residue contacts that is almost absolutely conserved. These CC conserved hydrophobic residues form only 10-15% of all the residues in the individual structures.A similar analysis of the cytochromes c'-b(562), which bind haem and have a very different function to that of the cytokines, gave very similar results. Again some 30% of the CC residues have hydrophobic residues with strong or medium conservation. Most of these form the buried spine of each helix and play the same role as those in the cytokines. The others, and some spine residues bind the haem co-factor.